A proteomic and genomic approach to in vivo chemoresistance using spheroid and xenograft cancer models [Elektronische Ressource] / vorgelegt von Lilja Thoenes

Thoenes, Lilja

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Dissertation
zur Erlangung des Doktorgrades der Fakultät für Chemie und
Pharmazie der Ludwig-Maximilians-Universität München



A proteomic and genomic approach to in vivo chemoresistance
using spheroid and xenograft cancer models

vorgelegt von
Lilja Thoenes
aus Starnberg
2009 Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 der Promotionsordnung
vom 29. Januar 1998 von Herrn Prof. Dr. Ernst Wagner betreut.










Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.

München, am 23.6.2009

…………………………...
(Lilja Thoenes)









Dissertation eingereicht am 23.6.2009
1. Gutachter: Prof Dr. Ernst Wagner
2. Gutachter: Prof Dr. Christian Wahl-Schott
Mündliche Prüfung am 21.07.2009Table of Contents
Table of Contents
1.  Introduction............................................................................................................ 1 
1.1.  Colon cancer ................................................................................................... 1 
1.1.1.  Low passage colon cancer cell lines ........................................................ 1 
1.1.2.  Threedimensional culture systems........................................................... 1 
1.1.3.  Proteomic profiling of multicellular spheroids of low passage colon
carcinoma cells......................................................................................... 2 
1.1.4.  Chemoresistance to 5-fluorouracil in colon cancer .................................. 4 
1.1.5.  Chemoresistance of low passage colon carcinoma cells towards 5-
fluorouracil treatment in vitro .................................................................... 4 
1.2.  Prostate Cancer .............................................................................................. 6 
1.2.1.  Metronomic Cyclophosphamide Therapy................................................. 6 
1.2.2.  Chemoresistance to antiangiogenic therapy ............................................ 7 
1.2.3.  Proteomics and genomics in drug resistance .......................................... 9 
1.2.4.  In vivo resistance of prostate cancer xenografts towards metronomic
cyclophosphamide therapy..................................................................... 10 
1.3.  Aim of this thesis ........................................................................................... 12 
2.  Materials and Methods........................................................................................ 14 
2.1.  Cell Biology Methods..................................................................................... 14 
2.1.1.  Cell culture.............................................................................................. 14 
2.1.2.  Multicellular spheroid culture.................................................................. 14 
2.1.3.  Oxygen and serum deprivation............................................................... 15 
2.1.4.  In vitro chemotherapy ............................................................................. 15 
2.1.5.  Cell viability, Apoptosis and Proliferation ............................................... 15 
2.1.6.  TUNEL Assay ......................................................................................... 16 
2.1.7.  Transfections .......................................................................................... 16 
2.1.8.  Immunocytochemistry............................................................................. 17 
2.1.9.  Flow cytometry and microscopy ............................................................. 18 
2.2.  Protein analysis ............................................................................................. 19 
2.2.1.  Bradford Assay ....................................................................................... 19 
2.2.2.  Westernblot............................................................................................. 19 
2.2.3.  2D-DIGE electrophoresis and mass spectrometry................................. 20 
2.3.  RNA analysis................................................................................................. 23 Table of Contents
2.3.1.  RNA-extraction and concentration measurement for RT-PCR .............. 23 
2.3.2.  RT-PCR .................................................................................................. 23 
2.3.3.  Micro Array Analysis............................................................................... 24 
2.4.  Ex vivo/ in vivo experiments.......................................................................... 27 
2.4.1.  Animals ................................................................................................... 27 
2.4.2.  Tumor models......................................................................................... 27 
2.4.3.  Tumor Measurement .............................................................................. 27 
2.4.4.  Chemotherapy ........................................................................................ 28 
2.4.5.  Tumor Histology...................................................................................... 28 
2.4.6.  Isolation of tumor/organs........................................................................ 29 
2.4.7.  Reisolation of tumor cells ....................................................................... 29 
3.  Results................................................................................................................. 31 
3.1.  Colon cancer ................................................................................................. 31 
3.1.1.  Fragmentation of lamin A/C in multicellular spheroid cultures of low
passage colon cancer cells .................................................................... 31 
3.1.2.  5-FU chemoresistance in low passage colon cancer cells in vitro......... 35 
3.2.  Chemoresistance in metronomic cyclophosphamide therapy of prostate
cancer xenografts: an in vivo chemoresistance............................................ 39 
3.2.1.  Generation of an appropriate in vivo passaged control.......................... 39 
3.2.2.  Drug efflux activity of reisolated PC3 cells ............................................. 40 
3.2.3.  Proliferation of reisolated cells under hypoxic conditions ...................... 41 
3.2.4.  Reimplantation of reisolated PC3 cells................................................... 41 
3.2.5.  Expression of vascular markers and functionality of tumor blood flow in
reimplanted tumors................................................................................. 43 
3.2.6.  2D-DIGE analysis of reisolated in vivo resistant PC3-D3 and PC3-D4
cells versus PC3-wt cells........................................................................ 44 
3.2.7.  Validation of potential protein candidates............................................... 50 
3.2.8.  Microarray analysis of chemoresistant PC3 xenografts compared to non
resistant control tumors .......................................................................... 57 
4.  Discussion ........................................................................................................... 73 
4.1.  Colon cancer ................................................................................................. 73 
4.1.1.  Fragmentation of Lamin A/C in multicellular spheroid of low-passage
colon carcinoma cells ............................................................................. 73 
4.1.2.  Chemoresistance in low passage colon cancer cells............................. 76 Table of Contents
4.2.  In vivo chemoresistance of prostate cancer in metronomic cyclophosphamide
therapy........................................................................................................... 78 
4.2.1.  A non classical mechanism of chemoresistance in vivo ........................ 78 
4.2.2.  Proteomics and Genomics: two complementary approaches to in vivo
resistance ............................................................................................... 80 
5.  Summary ............................................................................................................. 92 
6.  Appendix.............................................................................................................. 95 
6.1.  Tables............................................................................................................ 95 
6.2.  Abbreviations.........................................................................................